Compositions and methods for simultaneous detection of volatile sulfur compounds and polyamines

ABSTRACT

Disclosed are compositions and methods useful for the rapid and facile simultaneous detection of malodorous bacterial metabolites in samples of expired breath and other fluids. The invention enables estimation, by simple visual inspection and comparison against standards, of the concentration of polyamines and volatile sulfur compounds in the micromolar to millimolar range.

FIELD OF THE INVENTION

[0001] The invention relates to, among other fields, the field ofdiagnostic tests to detect the presence of pathogenic microorganisms.

BACKGROUND OF THE INVENTION

[0002] In normal circumstances many microorganisms live on or in humanswithout causing harm to these humans. However, an increased number ofcertain microorganisms can be associated with a disease state.Particularly problematic is an increase in the number of microorganismsthat produce significant concentrations of chemicals that arc toxic tohumans. Such toxins include: volatile sulfur compounds (VSCs), such ashydrogen sulfide, methyl mercaptan, dimethyl disulfide, and dimethylsulfide; and polyamines, such as putrescine and cadaverine. Higherconcentrations of volatile sulfur compounds and of polyamines areassociated with certain disease states and with socially stigmatizedconditions, such as halitosis, or bad breath. It would be desirable toenable facile simultaneous detection of volatile sulfur compounds andpolyamines in order to facilitate detection of the presence ofpotentially pathogenic microorganisms and of disease states andconditions with which these microorganisms are associated.

[0003] The principal VSCs produced by oral bacteria are hydrogen sulfide(H₂S) and methylmercaptan (CH₃SH). H₂S is produced by the bacterialenzyme L-cysteine desulthydrase, which degrades the amino acidL-cysteine to produce H₂S, ammonium and 2-ketopropanate. The other majorVSC, CH₃SH, is produced by the action of the bacterial enzymeL-methionine gamma lyase, which breaks down the amino acid L-methionineto produce CH₃SH, ammonium and 2-ketobutyrate. H₂S levels have beenreported to occur at levels as high as 2 mM in the gingival crevice ofpersons with severe periodontal disease.

[0004] In addition to VSCs, oral bacteria have also been shown toproduce high levels of polyamines; chief among these are1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine).Putrescine is produced by the action of the bacterial enzyme omithinedecarboxylase, which degrades the amino acid L-ornithine to produceputrescine and carbon dioxide. Cadaverine is produced by the bacterialenzyme lysine decarboxylase, which breaks down the amino acid L-lysineto cadaverine and carbon dioxide.

[0005] Millimolar levels of these malodorous bacterial waste productshave been reported in the gingival crevicular fluid (GCF) of personswith severe periodontal disease. Their presence imparts a foul odor tothe breath of persons with the disease. In addition to being malodorous,VSCs have also been shown to be toxic and adversely affect a number ofmammalian enzymes. Production of these waste products by bacteriaresiding on the dorsum of the tongue imparts a foul odor to expiredbreath, resulting in halitosis. Together these two classes of bacterialwaste products combine to impart a foul odor to the breath of personswith halitosis and periodontal disease. The levels of these wasteproducts in GCF correlate directly with the level of periodontal diseaseactivity. The levels of these waste products in expired air correlatedirectly with the level of bad breath or halitosis.

SUMMARY OF THE INVENTION

[0006] Accordingly, the invention provides compositions and methods forsimultaneous detection of volatile sulfur compounds and polyamines.

[0007] The invention provides inter alia a chromogenic (calorimetric)biochemical assay to detect volatile sulfur compounds (VSCs) andpolyamines produced by oral bacteria residing on the dorsum of thetongue and in the gingival crevice.

[0008] The invention provides inter alia an assay to detect VSCs andpolyamines in saliva and expired breath.

[0009] In an aspect of the invention, the absorbance of a chromogeniccomposition according to the invention correlates directly with theconcentration of VSCs and polyamines in a fluid with which thecomposition is intermixed.

DETAILED DESCRIPTION OF THE INVENTION

[0010] At the outset of the description, it is helpful to note themeaning of each of several terms recurring throughout the specificationand claims.

[0011] When used in connection with the invention, a “volatile sulfurcompound” or “VSC” refers to a composition having a thiol or —SHfunctional group or to a composition having a thiolate or sulfide anion.Examples of VSCs include hydrogen sulfide and methyl mercaptan(methanethiol).

[0012] When used in connection with the invention, “polyamine” has itsusual meaning in the biochemical arts and includes a composition of thegeneral formula NH₂RNH₂. Examples of polyamines include putrescine andcadaverine.

[0013] When used in connection with the invention, “DTNB” refers to5,5′-Dithiobis(2-nitrobenzoic acid). DTNB is also known as Ellman'sreagent and has CAS number 69-78-3.

[0014] When used in connection with the invention, “2-IT” refers to2-Iminothiolane. 2-Iminothiolane hydrochloride is known as Traut'sreagent and has CAS number 4781-83-3.

[0015] The invention provides inter alia a chromogenic (calorimetric)biochemical assay to detect volatile sulfur compounds (VSCs) andpolyamines produced by oral bacteria residing on the dorsum of thetongue and in the gingival crevice.

[0016] The invention provides inter alia an assay to detect VSCs andpolyamines in saliva and expired breath.

[0017] In an aspect of the invention, the absorbance of a chromogeniccomposition according to the invention correlates directly with theconcentration of VSCs and polyamines in a fluid with which thecomposition is intermixed.

[0018] In an embodiment, the invention provides a chemically stabilized,buffered composition comprising 5,5′-Dithiobis(2-nitrobenzoic acid)(DTNB) to detect VSCs and 2-Iminothiolane (2-IT) to detect polyamines.In this regard, DTNB and other so-called sulfhydryl reagents areexamples of VSC-reactive means, and 2-IT and other thiolating reagentswhich modify primary amino groups are examples of polyamine-reactivemeans.

[0019] In a particular embodiment, a composition according to theinvention contains 0.5 millimolar (0.5 mM) DTNB and 0.5 mM 2-IT in 20 mMimidazole or 20 mM sodium phosphate, pH 7.0 buffer containing 0.1 mMmanganese chloride (MnCl₂). Such composition is referred to infra as“reagent mixture.” Without intending to be bound by theory, it isobserved that, in such an embodiment, such concentration of MnCl₂ andsuch buffer and such pH are associated with particularly favorablestability, such that the shelf-life of the reagent mixture is 12 monthsor longer when stored at room temperature. A similar embodiment, inwhich a composition according to the invention contains 0.1 mM cobaltchloride or 0.1 mM magnesium chloride instead of the aforementioned 0.1mM manganese chloride, also possesses particularly favorable stability.In this regard, imidazole buffer around pH 7, phosphate buffer around pH7, manganese chloride, magnesium chloride, and cobalt chloride areexamples of shelf-life-enhancing means. Theory notwithstanding, it isnoted that magnesium is an alkaline earth metal and that managese andcobalt are transition metals.

[0020] Without intending to be bound by theory, it is known in the artthat DTNB reacts with the sulfide or thiolate anion formed from the —SHfunctional group present in H₂S or CH₃SH. In such reaction, one of thedisulfide sulfurs of DTNB is attacked by the HS⁻ or CH₃S⁻ anion,whereupon the 5-thio-2-nitrobenzoate anion (TNB) is released. TNB existsin equilibrium with the tautomeric thioquinone. The thioquinone has anabsorption (absorbance) maximum at 412 nm with a molar extinctioncoefficient of 13,600 M⁻¹ cm⁻¹. This allows for the reaction to bequantified spectrophotometrically or visually by the increase in theyellow color of the mixture.

[0021] While in the art the calorimetric reaction of DTNB may beregarded as optimally performed at pH 8.0, DTNB is unstable when storedfor several days at room temperature under such conditions. It wasunexpectedly found that the inclusion of metal salt and the adjustmentof pH around neutrality impart the favorable property of a markedlyextended shelf-life. By way of example and not of limitation, it wasfound that a DTNB mixture was maintained stable for more than one yearat room temperature when the mixture included phosphate or imidazolebuffer, pH 7.0, and 0.1 mM MnCl₂. Hence the invention provides interalia a chromogenic composition the absorbance of which correlatesdirectly with the concentration of VSCs in a fluid with which thecomposition is intermixed and which composition has a shelf-life of atleast one year.

[0022] It is known in the art that 2-IT thiolates primary amines underappropriate conditions. A composition according to the invention isuseful for the detection of primary amines, including polyamines, suchas the malodorous diamines putrescine and cadaverine. When a compositionaccording to the invention is intermixed or otherwise contacted with asample that contains diamines, 2-IT of the composition reacts withprimary amino (—NH₂) groups of the sample's diamines to yield a reactionproduct with at least one free sulfhydryl group. Such a free sulfhydrylgroup, or its thiolate anion, reacts with DTNB of the reagent mixture,releasing the TNB anion and causing an increase in absorbance at 412 nm.

[0023] Compositions and methods according to the invention are usefulfor detecting the presence of malodorous toxins produced by bacteriaresiding on the dorsum of the tongue and in GCF obtained from thegingival sulcus.

[0024] For example, in an aspect of a method according to the invention,tongue scrapings are obtained by gently rubbing the cotton tip of asterile, cotton-tipped applicator against the dorsum of the tongue toform a scraping-laden applicator tip. The scraping-laden applicator tipis then contacted for 2 minutes at room temperature with a volume of 0.1ml of reagent mixture contained within a sterile, screw-capped 1.5 mlmicrocentrifuge tube, forming a chromogen-developed applicator tip. Thecolor of the chromogen-developed applicator tip is compared to standardson a color-coded chart. The chart is scaled in shades of yellow from LOW(light yellow) corresponding to <0.2 mM H₂S to HIGH (bright yellow)corresponding to >2 mM H₂S.

[0025] Also by way of example, in an aspect of a method according to theinvention, for detecting the presence of VSCs and polyamines in GCF, aGCF sample is obtained by inserting a sterile paper absorbant point intothe gingival sulcus and waiting 1 minute for the GCF sample to absorb,thereby forming a GCF-laden paper point. The GCF-laden paper point isthen contacted for 5 minutes at room temperature with a volume of 0.1 mlof reagent mixture contained within a sterile, screw-capped 1.5 mlmicrocentrifuge tube, forming a chromogen-developed paper point. Thecolor of the chromogen-developed paper point is compared to a standardcolor chart calibrated linearly from 0 (none detectable) to 5 (extreme),corresponding to 2 mM hydrogen sulfide.

[0026] Also by way of example, in an aspect of a method according to theinvention, VSCs and polyamines are detected and quantifiedspectrophotometrically through use of the reagent mixture. In amicrocentrifuge tube, a GCF-laden paper point is submersed in 450microliters of 20 mM Tris-HCl, pH 7.75 buffer. When it is desired toperform a spectrophotometric assay according to the invention, a volumeof 50 microliters of a 10× concentrated reagent mixture in 50% DMSO, 50mM imidazole, pH 6.4 buffer containing 1.0 mM MnCl₂ is added to andintermixed with the contents of the microcentrifuge tube, and theresultant mixture is incubated at room temperature for 8 minutes.Afterwards, the incubated resultant mixture is transferred to asemi-micro 1.5 ml disposable plastic cuvet, for example by means of adisposable transfer pipette. The concentration of VSC and polyamines isdetermined by measuring the absorbance of the incubated resultantmixture through use of a spectrophotometer or colorimeter and bycomparison of the measured absorbance to a reference standard. Bymeasuring the absorbance of a negative control or a blank, backgroundabsorbance readings are obtained for use in comparison to the measuredabsorbance of the incubated resultant mixture.

[0027] A method of detection of VSCs and polyamines in GCF according tothe invention, such as described in either of the two immediatelypreceding paragraphs, is observed to possess a lower limit of detectionof no more than about 0.02 mM H₂S in a sample, and an upper limit ofresponsive range near about 2 mM H₂S. If the sample contains polyaminesbut essentially no VSCs, this 0.02 mM lower limit corresponds to aconcentration of no more than about 0.02 mM polyamines in the sample. Ifthe sample contains VSCs but essentially no polyamines, this lower limitcorresponds to a concentration of no more than about 0.02 mM VSCs in thesample. For detection of polyamines and VSCs according to the invention,a mode of carrying out the invention whereby a composition according tothe invention is prepared using, as solvent, reagent grade nanopurewater, wherein concentrations of adventitious metals are extremely low,is in general to be preferred.

[0028] The invention also provides methods for detection of VSCs andpolyamines in saliva and expired breath.

[0029] For example, in an aspect of a method according to the invention,for saliva testing, a filter paper disk is impregnated with the reagentmixture by contacting the disk with the reagent mixture, and allowingexcess solvent to evaporate, to form an impregnated disk. Theimpregnated disk is affixed to the bottom interior of a sterile,graduated specimen cup. A volume of saliva is then spit by a subjectonto the impregnated disk in the specimen cup. Reaction is allowed toproceed in and on the impregnated disk. After a fixed period of time,the concentration of VSCs and polyamines is determined by comparison ofthe color developed on the surface of the impregnated disk against astandard such as a scaled color chart.

[0030] For example, in an aspect of a method according to the invention,for breath testing, a volume of 0.1 ml of reagent mixture is contactedwith a filter paper disk, and excess solvent is allowed to evaporate, toform an impregnated disk. The impregnated disk is then affixed to thedistal aperture of a cylindrical tube, said tube having an aperture ateach end, the proximal aperture being of such length and width, forexample, that a subject might conveniently expire breath through theproximal aperture, and the distal aperture being of such length andwidth, for example, that the impregnated disk covers the entire distalaperture. A subject then expires breath into the tube through theproximal aperture of the tube and toward the distal aperture of thetube, at least a portion of the expired breath contacting theimpregnated disk affixed to the distal aperture of the tube. Reaction isallowed to proceed in and on the impregnated disk. After a fixed periodof time, the concentration of VSCs and polyamines is determined bycomparison of the color developed on the surface of the impregnated diskagainst a standard such as a scaled color chart.

[0031] While filter paper is an exemplary matrix according to theinvention, cotton is also a suitable matrix according to the invention,as is any of a number of other inert matrices, such as those derivedfrom polysaccharides which are low in primary amino groups.

[0032] When DTNB and 2-IT are included in a MnCl₂-stabilized bufferedmixture according to the invention, the levels of two principal classesof anaerobic bacterial waste products are conveniently detected andquantified either visually with a yellow color scale orspectrophotometrically with a colorimeter or spectrophotometer at 412nm. The simplicity of the use of compositions and methods according tothe invention facilitates in-office testing by health care professionalsand home testing by patients for halitosis and periodontal disease.Compositions and methods according to the invention thus are useful forthe detection of VSCs in samples comprising air, water, oil, or fluidsin contact with foodstuffs or foods, and hence are also useful for thedetection of products of microbial metabolism, pathogenicmicroorganisms, halitosis, VSC pollution, or food spoilage orcontamination.

[0033] The invention provides a diagnostic composition comprising 2-IT,DTNB, a solvent, a buffer, and a metal salt.

[0034] The invention provides a neutral diagnostic solution comprisingfrom about 0.1 to about 2.5 millimoles of 2-IT per liter of thesolution, from about 0.1 to about 2.5 millimoles of DTNB per liter ofthe solution, from about 0.02 to about 0.5 millimoles of MnCl₂ or MgCl₂or CoCl₂ per liter of the solution, and (a) from about 4 to about 100millimoles of imidazole per liter of the solution or (b) from about 4 toabout 100 millimoles of sodium phosphate per liter of the solution.

[0035] The invention provides a method of detecting the presence of aVSC or a primary amine in a sample of a physiological fluid, said methodcomprising the step of contacting the sample with a compositioncomprising 2-IT, DTNB, a solvent, a buffer, and a metal salt.

[0036] The invention provides a method of detecting the presence of aVSC or a primary amine in a sample of a physiological fluid, said methodcomprising the step of contacting the sample with a neutral diagnosticsolution comprising from about 0.1 to about 2.5 millimoles of 2-IT perliter of the solution, from about 0.1 to about 2.5 millimoles of DTNBper liter of the solution, from about 0.02 to about 0.5 millimoles ofMnCl₂ or MgCl₂ or CoCl₂ per liter of the solution, and (a) from about 4to about 100 millimoles of imidazole per liter of the solution or (b)from about 4 to about 100 millimoles of sodium phosphate per liter ofthe solution.

[0037] The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC.

[0038] The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein a neutral solution of the chromogen has ashelf-life of at least one year.

[0039] The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the color of the developed chromogen is yellow.

[0040] The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the chromogen comprises 2-IT and DTNB.

[0041] The invention provides a test kit comprising a chromogen, thecolor of which chromogen develops more rapidly after the chromogen iscontacted with a first mixture comprising at least 20 micromolarpolyamine or at least 20 micromolar VSC than the color develops in asecond mixture identical in composition to the first mixture except thatthe second mixture comprises no more than 1 micromolar polyamine and nomore than 1 micromolar VSC.

[0042] The invention provides a test kit comprising a chromogen, thecolor of which chromogen develops more rapidly after the chromogen iscontacted with a first mixture comprising at least 20 micromolarpolyamine or at least 20 micromolar VSC than the color develops in asecond mixture identical in composition to the first mixture except thatthe second mixture comprises no more than 1 micromolar polyamine and nomore than 1 micromolar VSC, wherein the test kit has a shelf-life of atleast one year.

[0043] The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC.

[0044] The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture that the second mixture comprises nomore than 1 micromolar polyamine and no more than 1 micromolar VSC,wherein the microorganism is an anaerobe or a facultative aerobe.

[0045] The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the physiological fluid comprises GCF, saliva,tongue scrapings, or expired breath.

[0046] It will therefore be readily understood by those persons skilledin the art that the invention is susceptible of a broad utility andapplication. Many embodiments and adaptations of the invention otherthan those herein described, as well as many variations, modificationsand equivalent arrangements, will be apparent from or reasonablysuggested by the present invention and the foregoing descriptionthereof, without departing from the substance or scope of the presentinvention. Accordingly, while the invention has been described herein indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the inventionand is made merely for purposes of providing a full and enablingdisclosure of the invention. The foregoing disclosure is not intended orto be construed to limit the invention or otherwise to exclude any suchother embodiments, adaptations, variations, modifications or equivalentarrangements, the invention being limited only by the claims appendedhereto and the equivalents thereof.

I claim:
 1. A chromogenic composition for intermixture or contactingwith a fluid in which fluid it is desired to determine the concentrationof VSCs and polyamines, said composition comprising a first reagent,said first reagent capable of reacting with a polyamine to form a VSC,and a second reagent, said second reagent capable of reacting with a VSCto form a product, said product possessing an absorption maximum in thevisible portion of the electromagnetic spectrum.
 2. A chromogeniccomposition useful for the detection of VSCs and polyamines, comprisingDTNB and 2-IT.
 3. A composition according to claim 2 and furthercomprising an alkaline earth metal or a transition metal.
 4. Acomposition according to claim 3 and further comprising a buffer.
 5. Acomposition according to claim 3, wherein the metal comprises manganese,magnesium, or cobalt.
 6. A composition according to claim 4, wherein thepH of the composition is between about 6.3 and about 7.7.
 7. Acomposition according to claim 6, wherein the pH of the composition isabout
 7. 8. A composition according to claim 7, wherein the shelf-lifeof the composition stored at room temperature is at least one year.
 9. Achromogenic composition useful for the detection of at least one of thegroup consisting of (a) a product of microbial metabolism, (b) apathogenic microorganism, (c) halitosis, (d) VSC pollution, and (e) foodspoilage or contamination, said composition comprising DTNB, 2-IT, ametal, and a buffer.
 10. A chromogenic composition useful for thedetection of VSCs, comprising DTNB, a buffer, and at least about 0.02millimoles of an alkaline earth metal or a transition metal per liter ofthe composition.
 11. A composition according to claim 10, wherein themetal comprises at least one of the group consisting of manganese,magnesium, and cobalt.
 12. A composition according to claim 10, whereinthe pH of the composition is about
 7. 13. A composition according toclaim 12, wherein the shelf-life of the composition is at least oneyear.
 14. A method of detecting VSCs and polyamines in a sample, saidmethod comprising the steps of contacting the sample with thecomposition of claim 1 to form a mixture, and comparing the colorintensity or absorbance of (a) the mixture to (b) a standard.
 15. Amethod according to claim 14, wherein the sample comprises aphysiological fluid.
 16. A method according to claim 15, wherein thephysiological fluid comprises GCF, tongue scrapings, saliva, or breath.17. A method according to claim 14, wherein the sample comprises atleast one of the group consisting of (a) air, (b) water, (c) oil, and(d) a fluid in contact with a foodstuff or a food.
 18. A method ofdetecting VSCs and polyamines in a sample, said method comprising thesteps of contacting the sample with the composition of claim 2 to form amixture, and comparing the color intensity or absorbance of (a) themixture to (b) a standard.
 19. A method according to claim 18, whereinthe sample comprises a physiological fluid.
 20. A method according toclaim 19, wherein the physiological fluid comprises GCF, tonguescrapings, saliva, or breath.
 21. A method according to claim 18,wherein the sample comprises at least one of the group consisting of (a)air, (b) water, (c) oil, and (d) a fluid in contact with a foodstuff ora food.
 22. A method of detecting microbial infection in or on asubject, said method comprising contacting a sample taken from thesubject with the composition of claim 1 to form a mixture, and comparingthe color intensity or absorbance of (a) the mixture to (b) a standard.23. A method of detecting microbial infection in or on a subject, saidmethod comprising contacting a sample taken from the subject with thecomposition of claim 2 to form a mixture, and comparing the colorintensity or absorbance of (a) the mixture to (b) a standard.
 24. Amethod of detecting one or more malodorous microbial metabolites in asample of saliva or breath of a subject, said method comprising thesteps of: contacting the composition of claim 1 with a matrix to form animpregnated matrix; contacting the sample with the impregnated matrix toform a mixed matrix; and comparing the color intensity or absorbance of(a) the mixed matrix to (b) a standard.
 25. A method of detecting one ormore malodorous microbial metabolites in a sample of saliva or breath ofa subject, said method comprising the steps of: contacting thecomposition of claim 2 with a matrix to form an impregnated matrix;contacting the sample with the impregnated matrix to form a mixedmatrix; and comparing the color intensity or absorbance of (a) the mixedmatrix to (b) a standard.
 26. A test kit useful for the detection of amicrobial metabolite, comprising the composition of claim
 1. 27. A testkit useful for the detection of a microbial metabolite, comprising thecomposition of claim
 2. 28. A composition comprising apolyamine-reactive means, a VSC-reactive means, and ashelf-life-enhancing means, wherein reaction of the polyamine-reactivemeans with a polyamine forms a product that reacts with the VSC-reactivemeans.
 29. A method of detecting a product of the metabolism of at leastone microorganism in a physiological fluid taken from a vertebrate, saidmethod comprising the step of contacting a sample of the physiologicalfluid with a composition comprising a polyamine-reactive means and aVSC-reactive means.
 30. A composition according to claim 28, wherein thepolyamine-reactive means comprises 2-IT, the VSC-reactive meanscomprises DTNB, and the shelf-life-enhancing means comprises at leastone of the group consisting of (a) a salt of at least one of the groupconsisting of manganese, magnesium, and cobalt and (b) a buffer.
 31. Amethod according to claim 29, wherein the physiological fluid is takenfrom the oral cavity of the vertebrate.
 32. A composition according toclaim 3, wherein the concentration of DTNB in the composition is about0.5 millimolar, the concentration of 2-IT in the composition is about0.5 millimolar, and the concentration of manganese chloride in thecomposition is about 0.1 millimolar.
 33. A method according to claim 29and further comprising the step of comparing (a) a color produced bycontacting the sample of the physiological fluid with the compositioncomprising the polyamine-reactive means and the VSC-reactive means to(b) a reference color chart.
 34. A method according to claim 29 andfurther comprising the step of measuring the absorbance obtained bycontacting the sample of the physiological fluid with the compositioncomprising the polyamine-reactive means and the VSC-reactive means. 35.A method according to claim 33, wherein the comparing enables a humanbeing comparing the color produced to the reference color chart toestimate the sum total of the concentration of VSCs and polyamines inthe sample.
 36. A method according to claim 34, further comprising thestep of comparing (a) the absorbance obtained by contacting the sampleof the physiological fluid with the composition comprising thepolyamine-reactive means and the VSC-reactive means to (b) theabsorbance obtained by contacting a negative control or a blank with thecomposition comprising the polyamine-reactive means and the VSC-reactivemeans.
 37. A matrix impregnated with the composition of claim
 1. 38. Amatrix impregnated with the composition of claim
 2. 39. A matriximpregnated with the composition of claim
 9. 40. A matrix impregnatedwith the composition of claim
 10. 41. A matrix impregnated with thecomposition of claim
 28. 42. A matrix impregnated with the compositionof claim 32.